Myosin X was cloned from a PCR screen for unconventional myosins in the frog inner ear and a full-length clone was subsequently obtained from a bovine smooth muscle library. Virtually nothing is known about its function, tissue distribution or subcellular localization. It appears to have binding sites for three light chains or calmodulin. We have engineered a heavy meromyosin (HMM)-like fragment that contains the head sequence and the short coiled-coil region predicted to dimerize. In addition, a FLAG epitope was engineered onto the carboxyl-terminus. This HMM was put into a baculovirus for expression in Sf9 cells. The cells were initially coinfected with the heavy chain containing virus as well as a virus that expresses calmodulin. The HMM could be purified by FLAG affinity chromatography. The HMM was well expressed as approximately 1-5 mg of HMM could be purified per liter of cell culture. Its MgATPase was greatly activated by actin and had a V-max of 1.5 s-1 and a K-m of 13 mM. However, the myosin X HMM did not support movement of actin filaments in the in vitro motility assay. By the second day following purification, the protein had aggregated. This is behavior similar to that of myosin II in which the essential light chain has been removed and suggested that myosin X perhaps contains light chains in addition to calmodulin. We then coinfected with a virus that expressed both the regulatory and essential light chain of nonmuscle myosin II. Myosin X HMM appeared to bind the regulatory light chain, but it sill did not move actin filaments and aggregated in a time-dependent manner. We are currently searching for the endogenous light chains.